Postharvest Biology and Technology 16 (1999) 139145

Ripening-related changes in ethylene production, respiration

rate and cell-wall enzyme activity in goldenberry (Physalis
peru6iana L.), a solanaceous species
Gustavo D. Trinchero
a
, Gabriel O. Sozzi
a,
*, Ana M. Cerri
b
,
Fernando Vilella
b
, Adela A. Fraschina
a
a
Departamento de Qu mica, Facultad de Agronom a, Uni6ersidad de Buenos Aires, A6da. San Mart n 4453,
1417 Buenos Aires, Argentina
b
Departamento de Produccion Vegetal, Facultad de Agronom a, Uni6ersidad de Buenos Aires, A6da. San Mart n 4453,
1417 Buenos Aires, Argentina
Received 22 July 1998; accepted 21 December 1998
Abstract
The ripening of goldenberry (Physalis peru6iana) is associated with a conspicuous climacteric rise in carbon dioxide
and ethylene production. Its respiration rate and ethylene biosynthesis can be classied as extremely high. Ethylene
yields between 7 and 24 nmol h
1
per g in the ripe/overripe stages thus compare favorably with production rates
previously reported for tomato fruit. As the fruit color turns from green (chlorophyll) to yellowish orange
(carotenoids) and a progressive softening occurs, several cell-wall enzyme changes arise. Pectinmethylesterase and a-
and b-galactosidase reach activity levels similar to those in tomato fruit. Pectinmethylesterase and a-galactosidase
increase toward the ripe stage. a-Arabinofuranosidase and b-glucosidase show lower activities but with an increasing
pattern during ripening. On the other hand, polygalacturonase and a-glucosidase activities are hardly noticeable.
1999 Elsevier Science B.V. All rights reserved.
Keywords: Physalis peru6iana; Goldenberry; Ripening; Ethylene; Respiration; Firmness; Pigments; Cell-wall enzymes
1. Introduction
Physalis peru6iana L., commonly known as
goldenberry or cape gooseberry, is a solanaceous
hairy plant native to tropical South America. This
perennial herb presents fuzzy, slender-pointed
heart-shaped leaves and yellowish owers. The
orange edible fruit, pleasantly avored (Berger et
al., 1989) and containing high levels of vitamin A,
B, C, carotene, phosphorus and iron (Hewett,
1993; Sarkar and Chattopadhyay, 1993), are glo-
bose seedy berries enclosed in an inated, blad-
der-like calyx or husk. The conspicuous postoral
growth and enlargement of the calyx lends itself
* Corresponding author. Tel.: +54-11-45248087; fax: +54-
11-45148739.
E-mail address: gsozzi@mail.agro.uba.ar (G.O. Sozzi)
0925-5214/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S0925- 5214( 99) 00011- 3
G.D. Trinchero et al. / Posthar6est Biology and Technology 16 (1999) 139145 140
as one of the best-known examples of persistent
sepals. The calyx takes over a certain defensive
function: berries with an intact expanded calyx,
containing air that reduces the specic weight, are
less prone to handling damage and have longer
storage life, probably due to both mechanical and
chemical protection (Baumann and Meier, 1993).
Goldenberry is a quick growing and high yield-
ing minor horticultural crop. It has achieved in-
creasing economic importance in the last two
decades and has been introduced as a specialized
culture in warm regions worldwide, particularly in
some American countries (USA, Mexico, Colom-
bia) as well as in specic areas of Oceania (Aus-
tralia and New Zealand), Asia (India) and Central
and South Africa (Klinac, 1986). The fruit may be
eaten fresh, in salads or in cocktails, or may be
used for preserves, sauces and pies (Klinac, 1986;
Hewett, 1993). Nowadays, this species is consid-
ered among the most promising crops for cool
subtropical areas of the USA (McCain, 1993).
Despite the increasing demand for this berry,
there is little or no information about its ripening
and postharvest characteristics.
Ripening of climacteric fruit such as tomato
(Lycopersicon esculentum Mill.) is mainly coordi-
nated by the biosynthesis of the gaseous phyto-
hormone ethylene which triggers major but still
only partially understood biochemical/physiologi-
cal processes such as color and avor develop-
ment and softening in texture. The breakdown of
middle lamella pectins is thought to be highly
inuential in the cohesiveness and weakening of
the cell-wall structure. It has been suggested that
ethylene regulates some enzymes, such as poly-
galacturonase and b-galactosidase II, responsible
for pectin hydrolysis (Sitrit and Bennett, 1998;
Sozzi et al., 1998a). The activities of other en-
zymes potentially acting on the hemicellulosic and
cellulosic cell-wall fractions have also been in-
volved in the ripening of tomato fruit (Fischer
and Bennett, 1991; Maclachlan and Brady, 1994).
Tomato fruit has been used as a model for
basic and applied ripening research (Brady, 1987).
In this study, we observed that goldenberry pre-
sents a similar climacteric evolution with interest-
ing differences in some ripening parameters. This
paper provides information on changes in
ethylene, CO
2
, rmness and pigments during gold-
enberry ripening. The presence of several enzymes
potentially involved in cell-wall turnover/solubi-
lization has also been examined.
2. Materials and methods
2.1. Plant material
Fruits of goldenberry (P. peru6iana L.) were
obtained from plants grown at the Facultad de
Agronom a, Universidad de Buenos Aires.
Seedlings were eld planted when they were 15
20 cm tall with at least 0.80 m between plants.
Plants were trellised but not fertilized. Fruits were
hand-picked using the following selection criteria,
modied from Baumann and Meier (1993): stage
IG: immature green berry, fresh calyx; stage MG:
mature yellowish green berry, greenish to pale
yellow calyx; stage Y: dark yellow berry, calyx not
completely dry; stage OR: yellowish orange ripe
berry, light brown, dry and paper-like calyx; stage
OO: yellowish orange overripe berry with mani-
fest softening. Fruit were harvested from the same
plants on March 12 (rst harvest) and 25 (second
harvest), 1998. Defect-free fruit of uniform shape
and size were washed in a 150 ppm chlorine
solution, rinsed with distilled water and air-dried.
2.2. Ethylene and carbon dioxide production
A closed static system was employed to deter-
mine ethylene and CO
2
production. For ethylene
measurement, the procedure involved enclosing
the fruit (12 cm in diameter; 24 g in weight) in
5-l tightly-sealed glass jars with 3 ml of 5 M KOH
to absorb evolved CO
2
and 5 ml of H
2
O to
conserve a high relative humidity. Gases were
allowed to accumulate for 1 h at 25C for yellow-
ish orange ripe and overripe berries, and 2 h at
25C for immature green, mature yellowish green
and dark yellow berries. Ethylene, O
2
and CO
2
production was monitored every 10 min. Oxygen
levels never dropped below 18% and CO
2
levels
remained below 0.05% in all containers. Ethylene
was determined by injecting 1 cm
3
of headspace
gas into a Hewlett Packard 5890 gas chro-
G.D. Trinchero et al. / Posthar6est Biology and Technology 16 (1999) 139145 141
matograph equipped with a ame ionization de-
tector and an activated alumina column kept at
90C, using nitrogen as the carrier gas. In all
cases, perfect linearity was observed during con-
nement. All measurements were replicated at least
twice.
CO
2
production was evaluated using 5-l jars,
performing gas extractions every 10 min. The jars
were kept in the dark to prevent photosynthesis.
Respiration rate (CO
2
production) was deter-
mined by taking 1 cm
3
of air from the jar and
injecting it into a portable infrared gas analyzer or
into a gas chromatograph equipped with a ther-
mal conductivity detector and molecular sieve 5A
and Porapack Q columns. CO
2
levels remained
below 0.2% in all cases and perfect linearity was
observed during 1-h connement. All measure-
ments were repeated at least three times.
2.3. Pigment e6aluation and rmness
For determination of total chlorophyll and
carotenoids, 1 g of frozen pericarp was extracted
ve times with 5 ml of acetone in a glass homoge-
nizer while covering the tubes with aluminium foil
to prevent photo-bleaching. The combined mix-
tures were nally extracted on a reciprocal shaker
at 140 rpm for 30 min. The acetone extract was
centrifuged at 12 000g for 15 min and brought
to a nal volume of 25 ml by adding acetone.
Chlorophyll and carotenoid content were deter-
mined as previously described (Lichtenthaler,
1987).
Texture measurements were made with an In-
stron Universal Testing Machine (Model 1011,
Canton, MA). Whole goldenberry fruit were
placed on a stationary steel plate. Each fruit was
punctured once at the equatorial axis. Puncture
tests involved use of a 4.8-mm at-ended cylindri-
cal probe on a drill base with a crosshead setting
of 50 mm min
1
. A total of 20 determinations per
maturity stage was obtained. Results were re-
ported in Newtons (N).
2.4. Enzyme extraction and assay
For enzyme extraction, all operations were con-
ducted at 4C. Enzyme solution was prepared
using 10- to 15-g composite samples of pericarp
tissue from 2230 different fruit for each replica-
tion. Polygalacturonase and pectinmethylesterase
were extracted as described by Gross (1982). Poly-
galacturonase was assayed using the 2-cyanoac-
etamide method (Gross, 1982) and
pectinmethylesterase by the spectrophotometric
procedure of Hagerman and Austin (1986). One
unit of polygalacturonase was dened as the
amount that catalyzed the formation of 1 mmol of
reducing sugars per min at 25C. Pectin-
methylesterase units were expressed in mmol
min
1
of galacturonic acid produced at 25C.
Glycosidases were extracted using a procedure
similar to that of Sozzi et al. (1998b). The tissues
were homogenized in 1 vol. (w/v) of cold 1.4 M
NaCl and pH was adjusted to 6 with 1 M NaOH.
The suspension was shaken for 1 h and squeezed
through cheesecloth. The ltrate was centrifuged
at 12 000g for 20 min. Extract aliquots were
desalted using PD-10 columns (Pharmacia Bio-
tech, Uppsala, Sweden). Aliquots of crude extract
were assayed for different activities by measuring
the release of p-nitrophenol from the correspond-
ing p-nitrophenyl glycoside (Sigma, St Louis,
MO). The reaction mixture consisted of 0.5 ml of
0.1 M citrate buffer, pH 5.3 for a-galactosidase
and a-arabinofuranosidase and pH 4 for b-galac-
tosidase, a-mannosidase and a- and b-glucosidase,
0.4 ml of 0.1% BSA, 0.1 ml of enzyme solution
and 0.4 ml of 13 mM substrate solution. Activity
determination was carried out by means of xed
time analysis. In all cases, the amount of p-nitro-
phenol released was found to be linear with time,
even over 60 min of incubation; a time period of
15 min was selected. After 15 min at 37C, the
reaction was stopped by adding 2 ml of 0.2 M
sodium carbonate. Absorbance was measured at
400 nm. One unit of each glycosidase was dened
as the amount that hydrolyzed 1 nmol min
1
of
p-nitrophenyl glycoside.
3. Results and discussion
Goldenberry is a climacteric-type fruit that dis-
plays a unique ripening pattern involving impres-
sive increases in ethylene and CO
2
production
G.D. Trinchero et al. / Posthar6est Biology and Technology 16 (1999) 139145 142
rates. Evaluation of goldenberry ethylene levels
showed a sharp 45-fold increase in this phytohor-
mone during ripening and a 70-fold increase when
the overripened stage was attained (Fig. 1). Gold-
enberry ethylene production rates reach extremely
high values in comparison with those of other
harvested commodities (Kader, 1992). Ethylene
production also showed signicant differences be-
tween harvest dates (Fig. 1). This may be at-
tributable to the variation in ambient temperature
conditions; fruit from the second harvest ripened
on the vine with higher ambient temperatures.
Nevertheless, other parameters did not vary sig-
nicantly between harvest dates.
An increase in respiration rate accompanies the
burst in ethylene production (Fig. 2). The golden-
berry fruit is somewhat unusual in that it contin-
ues to increase in weight throughout development
and ripening when still attached to the plant. This
weight increase is accompanied by a marked rise
in CO
2
production per fruit though the respira-
tion rate gradually drops between stages IG and
OR on a per-unit weight basis (Fig. 2). Color
changes are well correlated with chlorophyll
breakdown and carotenoid accumulation (chiey
b-carotene) in the plastids (Fig. 3).
Fig. 2. Carbon dioxide evolution in goldenberries harvested at
different developmental stages. Three replications of 616
fruit each were used for every measurement. Bars represent
S.D. of the mean. Where bars are not shown, the S.D. does
not exceed the size of the symbol.
Firmness values derived from puncture tests
reect the integrity of pericarp tissue (Holt, 1970),
where fruit softening enzymes are mainly local-
ized. Firmness decreased markedly during ripen-
Fig. 1. Ethylene evolution in goldenberries obtained at two
harvest dates and different developmental stages. Two replica-
tions of 2230 fruit each were used for every measurement.
Bars represent S.D. of the mean. Where bars are not shown,
the S.D. does not exceed the size of the symbol.
Fig. 3. Chlorophyll and carotenoid content in goldenberries
harvested at different developmental stages. Each point repre-
sents the mean9S.D. of three composite samples; at least
three fruit were used in each sample. Where bars are not
shown, the S.D. does not exceed the size of the symbol.
G.D. Trinchero et al. / Posthar6est Biology and Technology 16 (1999) 139145 143
Table 1
Firmness in goldenberry fruit picked at various stages of
ripeness
Developmental stage Puncture force (N)
a
18.4593.11 Mature yellowish green berry
Dark yellow berry 11.1191.38
Yellowish orange ripe berry 6.4690.74
Yellowish orange overripe berry 3.4290.32
a
Values shown are means9S.D. of 20 fruit for each devel-
opmental stage. In all cases, means are signicantly different
as determined by Tukeys test (PB0.05).
The relatively low polygalacturonase activity in
goldenberry supports the concept of glycosidase
involvement in cell-wall hydrolysis. From the six
assayed glycosidases, a- and b-galactosidases
showed the highest activity though only a-galac-
tosidase increased consistently with ripeness (Fig.
4A). A possible relationship between one b-galac-
tosidase isoenzyme (b-galactosidase II) and a
physiological function has been hypothesized for
Fig. 4. Enzyme activity in goldenberry at different stages of
development. (A) pectinmethylesterase, a-mannosidase, a- and
b-galactosidase activity; (B) a-arabinofuranosidase, a- and b-
glucosidase activity. Values represent the mean9S.D. of three
separate tissue preparations; 2230 fruit were used in each
sample. Where bars are not shown, S.D. does not exceed the
size of the symbol.
ing (Table 1), showing a signicant fruit deterio-
ration rate. Firmness of yellowish orange ripe
berries was about 35% that of mature yellowish
green berries. Fruit with scores below 4.5 N were
judged to be not marketable.
Ethylene is known to bring about promotive
effects on the softening of climacteric fruit; this
partially occurs as a consequence of cell-wall
weakening caused by the activity of different hy-
drolases (Fischer and Bennett, 1991; Oeller et al.,
1991; Sitrit and Bennett, 1998; Sozzi et al., 1998a).
Polygalacturonase activity was drastically lower in
goldenberry in all stages examined compared to
previous data in normal tomato pericarp (e.g.
Biggs and Handa, 1989). Polygalacturonase was
absent in the IG stage and did not exceed 0.006 U
g
1
during ripening. These levels are similar to
those observed in some ripening-impaired tomato
mutants (Biggs and Handa, 1989). Nevertheless,
there is strong evidence that fruit softening is not
regulated exclusively by polygalacturonase.
Pectinmethylesterase, a second pectolytic en-
zyme, was found to be present in goldenberry and
its activity gradually increased as ripening pro-
ceeded (Fig. 4A). Changes in pectinmethylesterase
activity also occur during tomato ripening
(Tucker et al., 1982; Gaffe et al., 1994). Neverthe-
less, transgenic tomatoes containing an antisense
pectinmethylesterase gene, the expression of
which leads to a 10-fold reduction in said enzyme
activity, show a marked loss in tissue integrity
during senescence but little modication on fruit
rmness during ripening (Tieman and Handa,
1994).
G.D. Trinchero et al. / Posthar6est Biology and Technology 16 (1999) 139145 144
tomato as it has been found to be capable of
catalyzing the in vitro degradation of a (14)-b-D-
galactan isolated from tomato cell walls (Pressey,
1983; Carey et al., 1995). Ethylene brings about a
promotive effect on b-galactosidase II (Sozzi et al.,
1998a) which reaches peak values at the ripe stage
(Pressey, 1983; Carey et al., 1995; Carrington and
Pressey, 1996; Sozzi et al., 1998a). Therefore, the
presence of a ripening-related b-galactosidase
isoenzyme may be masked in goldenberry crude
extract. a-Arabinofuranosidase and b-glucosidase
showed lower activity but with an increasing pat-
tern towards the ripe stage (Fig. 4B). a-Arabinosi-
dase presence may be related to the loss of
arabinosyl residues from fruit cell wall (Gross and
Sams, 1984). a-Mannosidase displayed maximum
activity during the immature stage (Fig. 4A) while
extracted a-glucosidase showed trace levels
throughout development and ripening (Fig. 4B).
Postharvest management of goldenberry, seek-
ing to maintain optimal quality levels, requires a
knowledge of the physiological and biochemical
mechanisms involved in its ripening process. This
work has determined some special characteristics in
goldenberry ripening; among them, the impressive
increase in ethylene biosynthesis and its high respi-
ration rate. These peculiarities are responsible for
goldenberry deterioration, as perishability of har-
vested fruits and vegetables is generally propor-
tional to the respiration rate. In goldenberry, the
use of modied or controlled atmospheres should
be considered as an interesting alternative to reduce
respiration and ethylene production, maintain
rmness and delay pathological decay. Evidence
about the pattern of some enzymes potentially
involved in cell-wall metabolism during ripening
has also been obtained. As far as we know, this is
the rst report on goldenberry ripening behavior.
In the context of the potential use of this species
as an alternative production, we feel that the
physiology of goldenberry ripening deserves fur-
ther research.
Acknowledgements
G.O. Sozzi fullls the requirements for the Doc-
toral degree at the Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires. This re-
search was supported in part by grants from the
Universidad de Buenos Aires to A.A. Fraschina
and F. Vilella (UBACyT Program).
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